Poxviruses. The cowpox virus is a member of the poxviridae, the poxvirus family, a large group of DNA-containing animal viruses. The classification and nomenclature of the poxviruses is described by Matthews, Intervirology 17: 42-46 (1982). The disclosure of this reference and all others cited in the background information and in the discussion of the invention are incorporated by reference herein. The poxviridae comprises two subfamilies: the chordopoxvirinae (poxviruses of vertebrates) and the entomopoxvirinae (poxviruses of insects). The poxviruses all have many similar structural, enzymatic, and genetic properties. They all replicate in the cytoplasm within viral "factories" (Cairns, Virology 11: 603-623 (1960)), also termed B-type cytoplasmic inclusions (Kato et al., Biken's J. 2: 353-363 (1959)).
In addition to the B-type cytoplasmic inclusions, many poxviruses produce large proteinaceous cytoplasmic inclusion bodies. Examples of chordopoxvirinae producing such inclusions (referred to as A-type inclusion bodies or ATIs) include the following: (1) Genus orthopoxvirus, ectromelia virus (Marchal, J. Pathol. Bacteriol. 33: 713-728 (1930)); cowpox virus (Downie, J. Pathol. Bacteriol. 48: 361-379 (1939)); (2) Genus parapoxvirus: bovine pustular stomatitis virus (Naginton, Vet. Rec. 82: 477-482 (1968)); (3) Genus avipoxvirus: fowlpox virus and canary poxvirus (Kato and Cutting, Stanford Med. Bull. 17: 34-45 (1959)); (4) Genus capripoxvirus: goatpox virus (Tantawi and Al Falluji, Acta Virol. 23: 455-460 (1979)); and (5) Genus suipoxvirus: swinepox virus (Teppema and DeBoer, Arch. Virol. 49: 151-163 (1975)). The possibility that members of the genus leporipoxvirus (rabbit myxoma and rabbit fibroma viruses) produce ATIs is discussed by Kato and Cutting, Stanford Med. Bull. 17: 34-45 (1959). Examples of entomopoxvirinae that produce proteinaceous cytoplasmic inclusion bodies (in addition to the B-type inclusions) are described by Bergoin and Dales, in Comparative Virology, eds. K. Maramorosch and E. Kurstak, 169-205, Academic Press, New York and London (1971).
Of all the poxviruses, those that have been studied most are those belonging to the genus orthopoxvirus (reviewed by Moss, Virology, ed. B. N. Fields, 685-703, Raven Press, New York (1985)). This genus includes vaccinia virus (the type species of the genus), cowpox virus, ectromelia virus, monkeypox virus, variola virus and raccoonpox virus. Studies on these viruses have provided the following general information on poxviruses.
The Expression of Poxvirus Genes. The DNA of vaccinia virus is about 180 kb long. It contains about 100 genes that are expressed before the onset of viral DNA replication ("early" genes), and it contains over 50 genes that are expressed after the onset of viral DNA replication ("late" genes). In addition to this temporal regulation of gene expression, the level of expression of each gene is regulated. The mechanisms that effect the temporal and quantitative regulation of expression of viral genes are poorly understood.
The transcription of each viral gene is tightly regulated, and this provides one mechanism of controlling viral gene expression. Viral proteins and specific viral cis-acting elements are required to effect the transcription of poxvirus genes (Puckett and Moss, Cell 35: 441-448 (1983); Cochran et al., Proc. Natl. Acad. Sci. USA 82: 19-23 (1985)), but little is known about them.
The early genes of vaccinia virus appear to have transcriptional promoter elements that comprise 30-40 bp upstream of the transcriptional start-site (Cochran et al., J. Virol., 54: 30-37 (1985); Weir and Moss, Virology 158: 206-210 (1987)). These elements do not resemble the transcriptional control elements of the genes of eukaryotes. The transcription of each gene is terminated 50-70 bp downstream of a thymidine-rich region in the non-transcribed strand of the viral DNA (Rohrman et al., Cell 46: 1029-1035 (1986)). Thus, the mRNAs of the early genes are a defined length. In addition, each is capped at its 5'-end, and each contains a poly(A)(adenine) tail of about 100 residues at its 3'-end.
The late genes of vaccinia virus do not appear to have transcriptional promoter elements that are similar either to those of the early genes, or to the genes of eukaryotes. The putative promoter elements of those late genes that have been characterized appear to be short (15-30 bp) sequences located immediately upstream of the end-point of complementarity between the mRNA and the DNA template (Cochran et al., J. Virol 54: 30-37 (1985)). This end-point may correspond to both the 5'-end of the mRNA and the transcriptional start-site. Alternatively, there is some evidence that the mRNAs of at least a few late genes contain either 5'-terminal poly(A) sequences or both poly(A) and additional nucleotide sequences at the 5'-end that are not complementary to the corresponding region of the template strand of the viral DNA (Bertholet et al., Cell 50: 153-162 (1987); Schwer et al., Cell 50: 163-169 (1987); Patel and Pickup, unpublished data). The unusual structures of the 5'-ends of these late mRNAs suggests that the transcription of poxvirus late genes might occur by a novel mechanism. The nature of this mechanism has not yet been determined.
The mRNAs of all previously characterized late genes of vaccinia virus also differ from mRNAs of early genes in that they do not appear to have defined 3'-ends and they are thus not uniform in length (Cooper et al., J. Virol. 37: 284-294 (1981); Mahr and Roberts, J. Virol. 49: 510-520 (1984); Weir and Moss, J. Virol. 51: 662-669 (1984)). The significance of differences between the early and late mRNAs. with respect to the structures of their 5'- and 3'-termini is currently unclear. One possible function of the structures of the termini of late mRNA is to enhance the stability of the mRNA. Both 5'- and 3'-end structures can affect the stability of an mRNA (see review by Brawerman, Cell 48: 5-6 (1987)). In addition, the sequence flanking the initiation codon in the mRNA may exert a strong influence on the efficiency with which that mRNA is translated (Kozak, Cell 44: 283-292 (1986)). Therefore, the structures at each end of an mRNA are likely to affect the amount of gene product synthesized from that mRNA.
Poxvirus-derived Expression Vectors. Animal viruses of several types, including the poxviruses, have been used to construct virus vectors that can direct the expression of cloned genes (for a review, see Rigby, J. Gen. Virology 64: 255-266 (1983)). Each virus vector system has its advantages and disadvantages, as discussed in more detail below.
Most of the work on poxvirus-derived vectors has been done with vaccinia virus. Several vaccinia virus vectors are currently in use; these vectors and their applications are reviewed by Mackett and Smith, J. Gen. Virology 67: 2067-2082 (1986). The patent of Paoletti and Panicali (U.S. Pat. No. 4,603,112) discloses methods of inserting cloned genes into the genome of vaccinia virus such that the virus can direct the expression of the cloned gene. Methods of inserting cloned genes into the genomes of large DNA-containing viruses are also described by Roizman and Lang, U.S. Pat. No. 4,554,159; Stunnenbe and Wittek, Eur. Pat. No. 198,328; Post and Roizman, Cell 25: 227-232 (1981); Mackett, Smith and Moss, Proc. Natl. Acad. Sci. USA 79: 7415-7419 (1982); Panicali and Paoletti, Proc. Natl. Acad. Sci. USA: 79: 4927-2932 (1982); and Mackett, Smith and Moss, J. Virol. 49: 857-864 (1984).
The advantages of poxvirus-derived expression vectors include the following:
1) Various poxviruses replicate in a wide variety of animals. Most importantly, many of them replicate in humans, other mammals or animals that are of economic importance. Some poxviruses such as vaccinia virus have a broad host range; they are capable of replicating in a variety of tissues in several different animals. This allows wide use of the vectors obtained from these poxviruses. Some of the other poxviruses are limited in their replication to certain hosts or to certain cell types. This may be a useful attribute, particularly when it is desirable to restrict a virus infection either to a specific animal or to specific cells. The fact that several poxviruses are able to replicate in localized regions of the skin of certain animals, without producing other serious effects on the animal provides a simple method of raising antibodies against the protein encoded by the cloned gene. Furthermore, this feature has stimulated the development of candidate, live, poxvirus-derived vaccines against several (non-poxvirus) pathogens. Most of these candidate vaccines are expression vectors derived from the strain of vaccinia virus used to vaccinate against smallpox virus. Each contains at least one gene cloned from the targeted pathogen. The aim is to construct virus vectors that will direct the synthesis of enough product of the cloned gene to stimulate the production of sufficient antibody against this protein to protect the inoculated animal from subsequent infection with the pathogen. If successful, humans and other animals might be immunized in this way against a variety of pathogenic agents. PA1 2) The genomes of most poxviruses contain large amounts of genetic information that may be experimentally replaced by other cloned genes. In addition, large amounts of DNA can be inserted within the genomes of poxviruses without any adverse effects. For example, Smith and Moss, Gene 25: 21-28 (1983), obtained a viable vaccinia virus recombinant whose DNA contained an insert of 25 kbp of phage lambda DNA. Therefore, unlike many of the other virus-derived expression vectors, the poxvirus-derived vectors are capable of containing relatively large inserts that might contain several cloned genes. PA1 3) Expression of the cloned gene may be delayed until late in the virus multiplication cycle. A cloned gene may be placed under the control of regulatory elements derived from those controlling the expression of a late viral gene. This may be useful if the product of the cloned gene is toxic to the cell. If the cloned gene is only expressed late, then some progeny virus will be produced before the cell is killed by the toxic gene product. This allows the production of some viable recombinant viruses containing the cloned gene, whereas the use of regulatory elements derived from an early gene might result in the production of gene products that kill the cell before viable recombinant viruses are produced. PA1 1) Most poxviruses produce a lytic infection resulting in the death of the infected cell. Consequently, the production of the protein encoded by the cloned gene is limited by the lifespan of the infected cell. PA1 2) Poxvirus-derived expression vectors that are currently in use have not been able to direct the synthesis of large amounts of protein. Thus, a significant advantage of this invention over the known prior art is that the vector of the invention provides a way to direct the synthesis of very large amounts of protein by selection of certain cis-acting elements of poxviruses. This is important if the object of using the system is either to recover the purified gene product or to revaccinate an animal that has already been immunized by means of a poxvirus-derived vaccine. The latter situation might arise either if a continued course of immunizations (over a period of months or years) is needed, or if multiple vaccinations are required with poxvirus-derived vectors directing the expression of different cloned genes.
The disadvantages of poxvirus-derived expression vectors for some uses include the following:
Not only are current poxvirus vectors subject to the disadvantage of low protein synthesis levels, but also few other eukaryotic virus expression vectors are able to direct high levels of expression of a cloned gene. Currently, in this regard, the most successful vector system is that of the baculovirus-derived expression vector system (see review by Doefler, Curr. Top. Microbiol Immunol. 131: 51-68 (1986)); Smith et a.l., Mol. Cell. Biol. 3: 2156-2165 (1983); Pennock et al; Mol. Cell. Biol. 4: 399-406 (1984)). Baculoviruses, which are not poxviruses, are viruses that replicate exclusively in insects. They produce crystalline proteinaceous inclusions bodies (polyhedra) in either the nuclei or the cytoplasm of the host cell. The use of the baculoviruses as expression vectors employing the transcriptional promoter element of the gene encoding the protein component of the nuclear inclusion bodies is disclosed in Smith and Summers, Eur. Pat. No. 127,839 and Miller, Eur. Pat. No. 155,476. Although these baculovirus-derived vector systems have achieved high levels of expression of several cloned genes, they have the disadvantage that this expression can only be obtained in cells derived from insects. Proteins produced in such cells might not be processed in the same way that they would be processed in mammalian cells. Furthermore, the baculovirus-derived vectors cannot be used, as can poxvirus-derived vectors, to raise antibodies against the product of a cloned gene. Therefore, although baculovirus-derived expression vectors are useful and capable of producing high levels of gene expression, they cannot provide some of the functions that poxvirus-derived expression vectors can, most notably, the ability to express cloned genes in mammalian cells.
As noted above, the genes of poxviruses appear to possess regulatory signals that are unlike those of genes of eukaryotes. Therefore, these signals cannot simply be replaced by strong eukaryotic promoter elements in order to gain high levels of gene expression. Instead, one option is to use control elements derived from a poxvirus gene that is strongly expressed. Usually, the most strongly expressed genes of a virus are the late genes many of which encode the major protein components of the virus particle. The putative promoter elements of some of these genes have been used in poxvirus-derived expression vectors (Mackett et al., J. virol 49: 857-864 (1984); Chakrabarti et al, Mol. Cell. Biol. 5: 3403-3409 (1985)), but the levels of gene expression obtained with these elements have been only moderate. The preferred embodiment of this invention utilizes one of the most strongly-expressed of all poxvirus genes, that encoding for the major protein component of the poxvirus A-type inclusion bodies (see below and Patel et al., Virology 149: 174-189 (1986), to gain high levels of expression of genes cloned into poxvirus-derived expression vectors. The vector of this invention is one in which the synthesis of cloned genes' mRNAs is placed under the control of the cis-acting elements derived from a gene directing the production of the mature mRNA encoding a poxvirus A-type inclusion protein.
A-type Inclusions. A-type inclusions or ATIs are large, well-defined, proteinaceous bodies, that are encoded by the viral genome and produced in the cytoplasm of cells late in the viral multiplication cycle of many poxviruses. Depending on the particular poxvirus strain, ATIs may or may not contain virus particles. In infected cell cultures, mature cowpox virus particles begin to appear at 4-5 hours after infection and readily detectable ATIs begin forming 8-9 hours after infection. Virus progeny are contained within the ATIs in some of the cowpox strains. ATIs are produced in almost all cells infected by cowpox viruses. Patel et al., Virology 149: 174-189 (1986) showed that the major component of ATIs produced in cells infected with cowpox virus is a protein having a molecular weight of about 160 kilodaltons (kDa). During the late stages of infection, this protein may comprise up to 4% of the total cellular protein but is not a part of the virus structure itself. This is an unusually large amount of a viral protein; most of the 100-200 viral proteins including the major structural proteins each comprise much less than 1% of the total cell protein. Consequently, they are not readily detectable against the background of cellular proteins when total proteins of infected cells are examined by Coomassie blue staining of proteins resolved by polyacrylamide gel electrophoresis. In contrast, the 160 kDa ATI protein of cowpox virus is easily detectable (Patel et al., Virology 14: 176-189 (1986).
Antibody raised against purified 160 kDa ATI protein of cowpox virus reacts specifically with the following abundant late proteins: a 155 kDa protein in cells infected with raccoonpox virus; a 94 kDa protein in cells infected with vaccinia virus; a 130 kDa protein in cells infected with ectromelia virus; and a 92 kDa protein in cells infected with monkeypox virus (Kitamoto et al., Arch. Virol. 89: 15-28 (1986); Patel et al. , Virology 149: 174-189 (1986); J. Esposito, D. Patel, and D. Pickup, unpublished results).
Therefore, representative orthopoxviruses of all types (with the possible exception of variola virus, which was not tested) direct the synthesis of a protein that is antigenically related to the 160 kDa ATI protein of cowpox virus. These proteins appear to be the products of genes related to the ATI gene (Esposito, Patel and Pickup, unpublished results). The smaller of these gene products (those of vaccinia virus and monkeypox virus) do not appear to aggregate into typical ATI structures.
Antibody directed against the purified 160 kDa ATI protein has not yet been used to probe the antigenic relatedness between the ATIs of the chordopoxvirinae and the proteins of the cytoplasmic inclusions produced by entomopoxviruses; however, it is noteworthy that Langridge and Roberts, J. Invert. Path. 39: 346-353 (1982) estimated the molecular mass of the protein component of an entomopoxvirinae cytoplasmic inclusion to be about 110 kDa, similar to the molecular mass of the ATI inclusion protein of cowpox virus.
The Gene Encoding the Major Protein Component of the A-Type Inclusions Produced by the Cowpox Virus. The major protein component of the ATIs produced by the Brighton red strain of cowpox virus is a 160 kDa protein (Patel et al., Virology 149: 174-189 (1986)). This protein appears to be one of the most abundant of all viral proteins in the cell. The gene encoding the 160 kDa ATI protein has been identified and characterized and has been designated the 160K gene (Patel and Pickup, manuscript submitted (1987)). This gene is contained within the Kpn I G fragment of the DNA of the Brighton red strain of cowpox virus (see FIG. 4). The transcribed portion of this gene is shown in this figure.
The nucleotide sequences at each end of the transcribed region of the 160K gene have been determined. The sequence containing the initiation codon of the 160K gene is shown in FIG. 1. This sequence contains the regulatory elements necessary to direct the transcription of the 160K gene. This upstream sequence from the 160K gene sequence will be referred to as cis-acting element I or CAE I. The most unusual feature of this sequence is the presence of 28 consecutive repeats of the triplet GAT (nucleotides 171-254). The significance of these repeats is currently unclear. The nucleotide sequence at the other end of the 160K gene, shown in FIG. 2, contains the nucleotide sequence corresponding to that at the end of the complementarity between the 3'-end of the 160K gene's mRNA and the template strand of the viral DNA (see FIG. 8). This downstream sequence from the 160K gene sequence will be referred to as cis-acting element II or CAE II.
Unlike the other characterized late mRNAs of poxviruses, which appear to have heterogeneous 3'-ends (Cooper et al., J. Virol. 37: 284-294 (1981); Mahr and Roberts, J. Virol. 49: 510-520 (1984); Weir and Moss, J. Virol. 51: 662-669 (1984)), the 160K gene's mRNAs are uniform in length.
In addition, some characterized late mRNAs of genes of vaccinia virus appear to have heterogeneous 5'-ends as well as heterogeneous 3'-ends. Bertholet et al., Cell 50: 153-162 (1987) have reported that the late mRNA of a late gene (11K gene) of vaccinia virus contains leader sequences that may be up to thousands of nucleotides long. These leaders appear to be derived from different regions of the viral genome and to be linked to the coding region of the late gene via a poly(A) sequence immediately upstream of the initiation codon. In contrast, Schwer et al., Cell 50: 163-169 (1987), reported that mRNAs of the same 11K gene contained only poly(A) leaders that were about 35 residues long.
The 5'-termini of the mRNA of the 160K gene of cowpox virus each contain a poly(A) leader sequence immediately upstream of the initiation codon. Most of these mRNAs contain leaders of between 5 and 20 A residues (Patel and Pickup, manuscript submitted, 1987). These 5'-terminal poly(A) sequences are not complementary to the corresponding region of the template strand of the viral DNA. It is not yet clear how these 5'-terminal poly(A) containing mRNAs are produced.
The function of the poly(A) leader sequence is also unknown. This structure may enhance the translational efficiency of the mRNA. Kozak (Cell 44: 283-292 (1986)) demonstrated that the presence of a purine residue three nucleotides upstream of an initiation codon would exert a dominant positive effect on the efficiency of translation from that initiation codon. The poly(A) tract provides a purine at the appropriate position. The effect of the remainder of the poly(A) leader sequence on translational efficiency is unknown.
Another possible function of the 5'-poly(A) leader might be to increase the stability of that mRNA. During the multiplication of vaccinia virus the cellular mRNAs are degraded at a greater rate than mRNAs in uninfected cells; however, the viral mRNA appears to be less susceptible than cellular mRNAs to the virus-induced RNA degradation (Rice and Roberts, J. Virol. 47: 529-539 (1983)). The 5'-terminal poly(A) sequences may contribute to the stability of the late viral mRNAs, thereby potentially increasing the pool of mRNA that is available for translation.
As noted above, the mRNAs of the 160K gene have one other unusual structural feature. Each has a defined 3'-end that corresponds to a position just downstream of the 160K gene's open reading frame (Patel and Pickup, manuscript submitted (1987)). It is not yet clear how these mRNAs+ defined 3'-ends are generated. They may be produced either by the termination of transcription at a specific site, or by RNA processing of some description. Whatever the mechanism, the sequence (CAE II) shown in FIG. 2 is sufficient to direct the production of defined 3'-ends. For example, when this sequence element was placed downstream of a cloned gene under the control of CAE I (see example VII), it directed the production of mRNAs that contained defined 3'-ends, whereas if the CAE II was absent, the 3'-ends of the mRNAs were heterogeneous in length.
The significance of the defined 3'-ends of the 160K gene's mRNAs is unknown. It is a feature that is unusual and perhaps unique among the late viral mRNAs. Therefore, it too may contribute to the unusually high level of expression of the 160K gene. Again, one possible mechanism by which it may exert such an effect is by increasing the stability of the mRNA, because the structure of the 3'-end of a mRNA can govern the rate at which that mRNA is degraded (for a recent review on the rate of decay of mRNAs, see Brawerman, Cell 448: 5-6 (1987)).
The use of a downstream cis-acting element inserted into a vector downstream of a cloned gene has not been found in the prior art. In this invention, use of a downstream cis-acting element derived from a gene encoding for a major A-type inclusion protein produces late mRNAs that have defined 3'-ends.
In summary, the 160K gene encoding the major protein component of the ATIs produced by cowpox virus appears to be one of the most strongly-expressed genes of this virus. This gene and its counterparts in the genomes of poxviruses of other types may prove to be the most strongly-expressed of all poxvirus genes. Accordingly, the features that effect such high levels of expression of these genes may provide the best means of directing high levels of expression of genes cloned into poxvirus-derived vectors. We have identified and characterized the cis-acting elements (CAE I and CAE II) that direct the synthesis of the mRNA of the 160K gene, and also determined the structures of the 5'- and 3'-ends of these mRNAs. We have modified these elements and used them to direct high levels of expression of genes cloned into poxvirus-derived vectors.